In response to high frequency action potential firing synapses exhibit extensive depression. This common form of synaptic plasticity brings computational advantage to synaptic circuits by increasing the sensitivity of neurons to subtle temporal changes in synaptic inputs. This depression is thought to be a direct outcome of the dynamics of vesicle recycling and vesicle depletion in presynaptic terminals. However, a clear mechanistic link between the dynamics of synaptic vesicle cycle and phases of synaptic depression is still lacking. The main goal of this project is to bridge this gap using a powerful combination of electrophysiology, optical imaging, electron microscopy and molecular biology in hippocampal synapses. Our studies have recently shown that synaptotagmin7 forms a molecular switch controlling the rate of vesicle recycling in a bi-directional manner through its alternative splice variants. This observation sets the stage for studies aimed at understanding the exact relationship between synaptic vesicle recycling and synaptic output. To fulfill this goal three specific aims are proposed. In the first specific aim, the regulation of vesicle recycling and synaptic depression by activity and second messengers, such as calcium and diacylglycerol, will be studied using fluorescent measurement of vesicle recycling and electrophysiology. In the second specific aim, the fast and slow recycling will be molecularly dissected through overexpression of synaptotagmin7 splice variants to examine their respective roles in regulation of presynaptic dynamics and neurotransmitter release. These functional experiments will be complemented by morphological analysis of synapse structure using electron microscopy. In the third specific aim, structural elements within synaptotagmin7 that control synaptic vesicle recycling will be identified through structure-function analysis in transfected synapses. These concerted investigations will enable us to understand with increasing precision the mechanistic link between recycling of synaptic vesicles and short-term synaptic plasticity. This information will also be critical for a better understanding of the pathologies underlying several neurological and psychiatric illnesses ranging from epilepsy to schizophrenia.